Composition for polymerizing immobilization of biological molecules and method for producing said composition

ABSTRACT

The invention refers to the field of molecular biology and bio-organic chemistry and deals with a composition for immobilization in polymer carriers of oligonucleotides, proteins, nucleic acids or any other molecules, which structure comprises active groups, including amino- or sulfhydryl groups, as well as a method for immobilization of various molecules including oligonucleotides, proteins, nucleic acids or any other molecules which structure comprises active groups, including amino- or sulfhydryl groups, consisting of a porous polymer being obtained on the basis of the composition claimed under conditions of addition or substitution reaction (radical, nucleophilic, or electrophilic nature, etc.) during synthesis of polymer by photo- and chemical initiated polymerization. 
     The composition claimed, as well as the method of immobilization of molecules in a polymer carrier using these compositions, may be used in various applications including microchips manufacturing that use is made of molecular biology on DNA sequencing and mapping, on detection of mutations and a whole range of medical applications.

FIELD OF INVENTION

The invention refers to the field of molecular biology and bio-organicchemistry and deals with a composition for immobilization of in polymercarriers of oligonucleotides, proteins, nucleic acids or any othermolecules, which structure comprises active groups, including amino- orsulfhydryl groups, as well as a method for immobilization of variousmolecules including oligonucleotides, proteins, nucleic acids or anyother molecules which structure comprises active groups, includingamino- or sulfhydryl groups, consisting of a porous polymer beingobtained on the basis of the composition claimed under conditions ofaddition or substitution reaction (radical, nucleophilic, orelectrophilic nature, etc.) during synthesis of polymer by photo- andchemical initiated polymerization.

The composition claimed, as well as the method of immobilization ofmolecules in a polymer carrier using these compositions, may be used invarious applications including microchips manufacturing that use is madeof molecular biology on DNA sequencing and mapping, on detection ofmutations and a whole range of medical applications.

BACKGROUND OF INVENTION

It is known in the art that cyclic compounds, or compounds containingtwo or more multiple bonds, are used for polymer synthesis by method ofradical polymerization, regardless of the method initiation thereof(photo-, thermal, radiation, or chemical initiation) [1].

[1] A. M.

,

(High-Molecular Compounds), M:

, 1981, c. 82-143.

The reaction proceeds by radical chain mechanism via initiation, chaingrowth, and chain termination steps. Such a nature of the radicalpolymerization provides favourable conditions for insertion in thepolymer structure of various molecules including oligonucleotides,proteins, nucleic acids, which structure comprises amino-, sulfhydryl orother active groups. There are at least two possible paths of insertion:

1. Molecules, which structure comprises amino-, sulfhydryl or otheractive groups, take part in chain transfer reaction to form aminyl,sulfanyl, or other active radicals. These radicals are capable to insertin the polymer structure both on chain growth step and on chaintermination step under intermolecular recombination [2,3,4].

[2] A. Good and J. C. Thynne, J. Chem. Soc., 1967, p. 684.

[3] C. J. Micheida and D. H. Campbel, J. Amer. Chem. Soc., 1976, 98, p.6728.

[4] C. J. Micheida and D. H. Campbel, Tetrahedron Letters, 1977, p. 577.

2. Molecules, which structure comprises amino-, sulfhydryl, or otheractive groups, take part in reaction of nucleophilic addition tounsaturated compounds [5] that present on various polymerization steps,namely with initial monomers, growing unsaturated macroradicals, andunsaturated macromolecules.

[5]

(General Organic Chemistry), T. (v.) 3,

(Nitrogen-Containing Compounds), H. K.

(Ed.), M:

, 1982, c. 61-62.

SUMMARY OF INVENTION

The essence of invention consists in that there are provided:

A composition for immobilization of molecules includingoligonucleotides, proteins, and nucleic acids, which structure comprisesactive groups including amino- and/or sulfhydryl groups in polymercarriers (polymers) under conditions of addition or substitutionreaction (radical, nucleophilic, electrophilic etc.) during synthesis ofpolymer by photo- and chemical initiated polymerization; and

A method for immobilization of molecules including oligonucleotides,proteins, and nucleic acids, which structure comprises active groupsincluding amino- and/or sulfhydryl groups in polymer carriers (polymers)under conditions of addition or substitution reaction (radical,nucleophilic, electrophilic etc.) during synthesis of polymer by photo-and chemical initiated polymerization.

The present composition and method of immobilization of variousmolecules in a polymer carrier using this composition will be used invarious applications including a microchip manufacturing.

For immobilization of molecules which structure comprises active groupsincluding amino- or sulfhydryl groups in polymer carriers, there isprovided a composition of the following formula:K=A ^(a) +B ^(b) +C ^(c) +D ^(d) +E ^(e) +F ^(f)

-   -   wherein    -   K is a composition;    -   A is acrylamide, methacrylamide,        N-[tris(hydroxymethyl)methyl]acrylamide,        2-hydroxyethylmethacrylate or the monomer based on derivatives        of acrylic, methacrylic, cinnamic, crotonic, vinylbenzoic or        other unsaturated acids;    -   B is N,N′-methylenebisacrylamide,        N,N′-1,2-dihydroxyethylenebisacrylamide, polyethyleneglycol        diacrylate, a mixture thereof or a symmetric or asymmetric        cross-linking agent based on derivatives of acrylic,        methacrylic, cinnamic, crotonic, vinylbenzoic or other        unsaturated acids;    -   C is an oligonucleotide, nucleic acid, protein or another        molecule bearing an active group including an amino- or        sulfhydryl group;    -   D is glycerol, sucrose, polyhydric alcohols, or another        high-boiling compound;    -   E is water, N,N-dimethylformamide, dimethylsulfoxide, and other        polar and non-polar solvents;    -   F is ammonium persulfate, potassium persulfate, methylene blue,        fluorescein, N,N,N′,N′-tetramethyl ethylenediamine, hydrogen        peroxide, 4-(N,N-dimethylamino)pyridine, triethylamine, acetone        or any other initiator for chemical or photo-initiated        polymerization.    -   a, b, c, d, e, f are percentages of any ingredient in the        composition (for solids X=m/v×100% and for liquids X=v/v×100%).    -   3≦a+b≦40%; 0≦c≦10%; 0≦d≦95%; 0≦e≦95%, 0≦f≦90%.

Along with polymers of another application, the composition as indicatedabove is proposed to use for manufacturing oligonucleotide, protein, DNA(RNA), and other biological microchips.

The present composition for manufacturing biological microchips willmake use of the following ingredients:

Monomers Composing the Basis of Polymer Carrier Being Formed

As monomers forming a linear polymer, there are to be used derivativesof acrylic, methacrylic, cinnamic, vinylbenzoic, crotonic or otherunsaturated acids including their esters and amides, for example,acrylamide, methacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide,2-hydroxyethylmethacrylate, methylmethacrylate etc.

As cross-linking agents for forming the three-dimensional gelstructures, there are used compounds, which structure comprises two ormore unsaturated fragments from which at least one is active in theaddition or substitution reactions (radical, nucleophilic,electrophilic, etc.), for example, N,N′-methylenebisacrylamide,N,N′-methylenebismethacrylamide, N,N′-1,2-dihydroxyethylenbisacrylamide, etc. or symmetric and asymmetric esters, amides, and mixedderivatives of acrylic, methacrylic, cinnamic, crotonic, vinylbenzoic,or other unsaturated acids.

Total content of monomer forming the linear polymer, (A), andcross-linking agent, (B), in the composition is in the range of 3-40%(3<T<40), and their ratio being in the range of 97:3-60:40% (3<C<40).

Polymers having a predetermined pore size are to be prepared by varyingthe combinations and ratios of monomers.

Oligonucleotides, Proteins, Nucleic Acids, which Structure ComprisesActive Groups Including Amino- and Sulfhydryl Groups

It is necessary for immobilization of oligonucleotides, nucleic acids,proteins or other molecules in the polymer carrier during its synthesison chemical or photochemical initiation that molecules being immobilizedcomprise active groups which are capable to go into addition orsubstitution reactions (radical, nucleophilic, electrophilic, etc.) withfragments of polymer being formed during its synthesis. In particular,amino-H sulfhydryl groups are provided as such active groups.

Oligonucleotides, which structure comprises amino- or sulfhydryl groups,are prepared under conventional conditions of phosphoramidite chemistryusing commercially available phosphoramidites.

DNA fragments having amino- or sulfhydryl groups are prepared underconditions of symmetric or asymmetric polymerase chain reaction using asynthetic primer bearing terminal amino- or sulfhydryl groups [6] or byamination of DNA fragments using a procedure as described in a reference[7].

[6] Yasunaga, S., Kimura, A., Hamaguchi, K., Ronningen, K. S., andSasazuki, T., Tissue Antigens., 1996, 47., 37-48.

[7] Proudnikov D, Timofeev E, Mirzabekov A., Anal Biochem. 1998, 15;259(1), P. 34-41.

For immobilization, protein molecules having free amino-, sulfhydryl,and other active groups make use of native form, without additionalmodifications.

In the compositions, the content of molecules having active groups (C)is varied in the range of 0≦c≦10%.

Medium Used for Immobilization of Molecules Having Amino- and SulfhydrylGroups in Polymer Carriers.

As a medium for a polymerizing immobilization of molecules, there areprovided water, aqueous solutions of polyvinyl alcohol, sucrose,dimethylformamide, dimethylsulfoxide or other polar water soluble andnon-polar compounds as well as anhydrous solutions of dimethylsulfoxide,dimethylformamide, formamide, etc. In the compositions, water content(E) is varied in the range of 0-95% (v/v), and a high-boiling compound(D) is of 0-95% (w/v; v/v).

Various compounds may be added in solutions for polymerization, forexample, glycerol to produce compositions of various viscosities thatallows varying a size of microchip gel elements under a pin robotdiameter fixed.

Initiators for Chemical and Photo-Initiation

To initiate the radical polymerization during synthesis of polymercarrier, there are proposed to make use of initiators and polymerizationpromoters soluble in water and organic media, namely: ammoniumpersulfate, potassium persulfate, hydrogen peroxide, ferrous salts,N,N,N′,N′-tetramethyl ethylenediamine, triethylamine, benzoyl peroxide,azobisisobutyric acid dinitrile, 4-(N,N-dimethylamino)pyridine, acetone,methylene blue, fluorescein, etc.

Monomers Covalently Attached to a Glass Surface

For modification of a glass surface with the purpose of covalent bondinga polymer carrier to the surface, there are provided the followingreagents: 3-trimethoxysilylpropyl methacrylate, 3-trimethoxysilylpropylmethacrylamide, 3-trimethoxysilylpropyl acrylamide, 3-glicidyloxypropyltrimethoxysilane.

Such a set of modifying agents makes it possible to form a reliablebonding a polymer to a glass over a broad range of medium pH (2-12) andof temperature (−10° C.-+100° C.).

Various combinations of composition ingredients allow obtaining polymercarriers which porosity may change over a broad range that makes itpossible to use these carriers in many applications, particularly forbiochip manufacturing.

An additional aspect of the present invention is a method forimmobilization of molecules including oligonucleotides, proteins, andnucleic acids, which structure comprises active groups consisting oflinear or three-dimensional porous polymer that is obtained on the basisof composition of formula:K=A ^(a) +B ^(b) +C ^(c) +D ^(d) +E ^(e) +F ^(f)

-   -   wherein:    -   K is a composition;    -   A is acrylamide, methacrylamide,        N-[tris(hydroxymethyl)methyl]acrylamide,        2-hydroxyethylmethacrylate or another monomer based on        derivatives of acrylic, methacrylic, cinnamic, crotonic,        vinylbenzoic or other unsaturated acids;    -   B is N,N′-methylenebisacrylamide,        N,N′-1,2-dihydroxyethylenebisacrylamide, polyethyleneglycol        diacrylate, a mixture thereof, or symmetric or asymmetric        cross-linking agent based on derivatives of acrylic,        methacrylic, cinnamic, crotonic, vinylbenzoic or other        unsaturated acids;    -   C is an oligonucleotide, nucleic acid, protein, or another        molecule bearing an active group including amino- or sulfhydryl        groups;    -   D is a glycerol, sucrose, polyhydric alcohol, or another        high-boiling compound;    -   E is water, N,N-dimethylformamide, dimethylsulfoxide or other        polar and non-polar solvents;    -   F is ammonium persulfate, potassium persulfate, methylene blue,        fluorescein, N,N,N′,N′-tetramethylethylene diamine, hydrogen        peroxide, 4-(N,N-dimethylamino)pyridine, triethylamine, acetone,        or any other initiator for chemical or photo-initiating        polymerization.    -   a, b, c, d, e, f are percentages of any ingredient in the        composition (for solids X=m/v×100% and for liquids X=v/v×100%).    -   3≦a+b≦40%; 0≦c≦10%; 0≦d≦95%; 0≦e≦95%, 0≦f≦90%.

For purposes of the present invention, the polymer is preferablyobtained by polymerization of (A+B) mixtures using combinations ofacrylamide, methacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide,N,N′-methylenebisacrylamide, N,N′-(1,2-dihydroxyethylenebisacrylamide,polyethyleneglycol diacrylamide, or other unsaturated compounds.

It is preferred where molecules including oligonucleotides, proteins,and nucleic acids, which structure comprises active groups includingamino- and/or sulfhydryl groups, react with fragments of a polymercarrier (polymer) being formed during its synthesis under conditions ofaddition or substitution reactions (radical, nucleophilic,electrophilic, etc.) on photo- or chemical initiated polymerization.

Moreover, oligonucleotides, which comprise active groups includingamino- and/or sulfhydryl groups, aminated DNA, DNA with sulfhydryl groupinserted as well as proteins, are not subjected to a modification beforethe immobilization process.

A method is further specified in that the immobilization of proteins isperformed in polymer carrier either by sulfhydryl groups, or byamino-groups, or by third functionality of aminoacids.

A method is further specified in that the immobilization ofoligonucleotides is performed in polymer carrier either by 5′-terminalof oligonucleotide or by 3′-terminal of oligonucleotide.

The method of present invention is further specified in that a polymerlayer formed is covalently bonded to a substrate or the covalent bondingto said substrate is absent.

In this case, the polymer layer formed on a substrate is athree-dimensional gel.

The polymer layer formed on a substrate is further a compact continuouslayer.

Additionally, the polymer layer as formed on substrate is divided byempty spaces into some cell and each cell will comprise (or notcomprise) macromolecules immobilized, and macromolecules beingimmobilized in various cells will have different nature and properties.

The cell as mentioned above forms the regular one- or two-dimensionalstructure (phase).

The application of the polymerization mixture on substrate is preferablycarried out by using an automatic device (robot) which is equipped withone or more microdispensers, and said microdispensers will be a pin typemicrodispensers or contactless microdispensers of jet type. Moreover,the method will make use of several microdispensers forming a regularstructure.

According to the present invention, the method envisages that beforepolymerization, one or more substrates including some droplets ofcomposition are hold at a sealed container which comprises the samecomposition mixture in amounts more than two times exceeding the totalamount of the polymerization mixture as supported on substrate.

Moreover, one or more substrates including supported droplets ofcomposition, before and during polymerization, are placed into thesealed container under oxygen free inert atmosphere with controlledhumidity.

According to the present method, said container is filled with one ofthe following gases: N₂, Ar, CO₂,

-   -   and gaseous medium being continuously or periodically restored        in the container with substrates.

BRIEF DESCRIPTION OF DRAWINGS

The invention is illustrated by using drawings wherein:

FIG. 1 shows the immobilization scheme for oligonucleotide having aterminal amino-group on a modified glass.

Oligonucleotide immobilized (C=130 pmol/μl):

Hybridized oligonucleotide labeled with fluorescent agent:

-   -   5′-CTCAGTTC-TexRed (1 μM, IM NaCl). Gels for immobilization:

A{methacrylamide:N,N′-methylenebisacrylamide-T5%, C5%};

B{acrylamide:N,N′-methylenebisacrylamide-T5%, C5%}.

FIG. 2 shows the immobilization of DNA bearing amino- or sulfhydrylgroups on a modified glass.

Gels for DNA immobilization:methacrylamide:N,N′-methylenebisacrylamide-T5%, C5%; DNA immobilized(C=1×10⁻³ g/mL): separated from a calf's thymus and subjected to anamination procedure by a protocol as disclosed in reference [4];

Hybridized oligonucleotide labeled with fluorescent agent:

5′-CTCAGTTC-TexRed (1 μM, IM NaCl). A and B are gels which do notcomprise or comprise DNA immobilized, correspondingly.

FIG. 3 illustrates an immunoassay on the microchip. The microchipcontains immobilized monoclonal antibodies to the green fluorescentprotein (Ab-GFP), to α-fetoprotein (Ab-AFP), to human immunoglobulin G(Ab-HIgG), and to human immunoglobulin G (HIgG). The antibodyconcentrations in the gel are as follows, μg/mL: 630 (series 1), 315(series 2), 210 (series 3), 130 (series 4), 60 (series 5), 40 (series6). Antibodies have been developed with the green fluorescent protein(a) and by antibodies to fluorescein labeled HIgG (b).

The invention will be further explained by using examples to illustratethe preferred embodiments of invention. These examples should not to beconstrued as a limitation of the scope of invention. Persons skilled inthe art will make numerous improvements which are also entered in thescope of claims of the present invention as set forth in the claims.

EXAMPLES Example 1 Immobilization of Oligonucletides ComprisingAliphatic Amino-Group in a Polymer Carrier

An aqueous solution of oligonucleotide (2.3 μL, C=1 nmol/μL) andglycerol (6.45 μL) are added to a solution of methacrylamide andN,N′-methylenebisacrylamide in 3M aqueous solution ofN,N,N′,N′-tetramethylethylene diamine (1.25 μL, 40% (m/v), 19:1). Themixture is thoroughly stirred. The solution is applied by using robot“GMS 417 Arraer” on a glass treated with Bind-Silane. A block ofdroplets obtained is exposed to ultra-violet rays (λ=312 nm, 30 min,T=55° C.) under dry argon atmosphere, washed with water (4 h, T=60° C.),and dried in a dust-free air (T=25° C.). This example makes use ofcomposition of the following formula: A—methacrylamide,B—N,N′-methylenebisacrylamide, C—oligonucleotide, D—glycerol, E—water,F—N,N,N′,N′-tetramethylethylene+diamine, a+b=5.00%, c=0.0006%; d=65.00%;e=20.00%, f=10.00%.

Oligonucleotide microchip obtained is used to perform a hybridization,PCR, etc.

FIG. 1 shows a result of hybridization on oligonucleotide microchip asobtained by the foregoing procedure.

Example 2 Immobilization of Aminated DNA in a Polymer Carrier

The procedure used is as set forth in example 1.

For immobilization, DNA is used as separated from a calf's thymus andaminated by a protocol as disclosed in reference [4]. DNA concentrationin the polymerization mixture is of C=1 mg/mL. This example makes use ofcomposition of the following formula: A—methacrylamide,B—N,N′-methylenebisacrylamide, C—DNA, D—glycerol, E—water,F—N,N,N′,N′-tetramethylethylene diamine, a+b=5.00%, c=0.00023%;d=65.00%; e=20.00%, f=10.00%.

FIG. 2 shows a result of hybridization on DNA-microchip as obtained bythe foregoing procedure.

Example 3 Immobilization of Protein in a Polymer Carrier

The procedure used is as set forth in example 1.

A solution of native form protein in a borate buffer (pH 8.3) is addedto a solution of polymerization mixture. The protein concentration inthe polymerization mixture is of C=630 μg/mL. The mixture is thoroughlystirred. The solution is applied by using robot “GMS 417 Arraer” on aglass treated with Bind-Silane. A block of droplets obtained is exposedto ultra-violet rays (λ=312 nm, 40 min, T=27° C.) under dry argonatmosphere. The protein microchips are further washed with a phosphatebrine buffer (0.01 M, pH 7.0) containing 0.1% Tween 20 then hold for 1 hin the phosphate brine buffer (0.01M, pH 7.0) containing both 1% BSA and5% of sucrose, and used for performing analyses of various types.

This example makes use of composition of the following formula:A—methacrylamide, B—N,N′-methylenebisacrylamide, C—DNA, D—glycerol,E—water, F—N,N,N′,N′-tetramethylethylene diamine, a+b=5.00%, c=0.0001%;d=65.00%; e=20.00%, f=10.00%.

FIG. 3 illustrates results of an immunoassay on the microchip.

1. A method for immobilizing oligonucleotide, nucleic acid and/orprotein molecules bearing an active aliphatic amino, an active aliphaticsulfhydryl group or both, in a polymer carrier said method comprisingreacting active groups of molecules with a radical of a polymer ormonomer during polymerization.
 2. A method according to claim 1, whereinthe polymer carrier is formed by polymerization of a compositioncomprisingK=aA+bB+cC+dD+eE+fF wherein K is the composition; A is a monomerselected from the group consisting of acrylamide, methacrylamide,N-[tris(hydroxymethyl)methyl]acrylamide, 2-hydroxyethylmethacrylate,methylmethacrylate, and other monomer based on derivatives ofunsaturated acids selected from the group consisting of acrylic,methacrylic, cinnamic, crotonic, and vinylbenzoic acid; B is a symmetricor asymmetric cross-linking agent based on derivatives of unsaturatedacids selected from the group consisting of acrylic, methacrylic,cinnamic, crotonic, and vinylbenzoic acid, orN,N′-methylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide,and polyethyleneglycol diacrylate, or a mixture thereof; C is abiological macromolecule selected from the group consisting ofoligonucleotide, nucleic acid, or protein bearing an active groupincluding an amino- or sulfhydryl group; D is a medium for performingthe polymerizing immobilization; E is a polar or non-polar solvent; F isa compound for promoting photo- or chemical initiated radicalpolymerization selected from the group consisting of ammoniumpersulfate, potassium persulfate, hydrogen peroxide, methylene blue,fluorescein, N,N,N′,N′-tetramethylethylenediamine,4-(N,N-dimethylamino)pyridine, triethylamine, and acetone; a, b, c, d,e, and f are percentages (X) of each ingredient in the compositionwherein for solids X equals m/v×100% and for liquids X equals v/v×100%;wherein the total amount of monomer A and cross-linking agent B iswithin the range of 3-40% (3≦a+b≦40%), wherein the monomer tocross-linking agent ratio being within the range of 97:3-60:40(3≦[b/(a+b)]≦40%); and percentages of ingredients C, D, E, and F beingwithin the ranges 0≦c≦10%, 0≦d≦95%; 0≦e≦95%, and 0≦f≦90%.
 3. The methodaccording to claim 1 wherein the polymerization is performed underconditions suitable for addition or substitution reactions.
 4. Themethod according to claim 1 wherein the polymerization comprises photo-or chemical initiated polymerization.
 5. The method according to claim2, wherein polymer is obtained by polymerization of a mixture of A+Bwherein A is selected from the group consisting of acrylamide,methacrylamide, and N-[tris(hydroxymethyl)methyl]acrylamide, and B isselected from the group consisting of N,N′-methylenebisacrylamide,N,N′-(1,2-dihydroxyethylene)bisacrylamide, and polyethyleneglycoldiacrylate.
 6. The method according to claim 2, wherein theoligonucleotides which contain active groups including amino- and/orsulfhydryl groups; aminated DNA; DNA with a sulfhydryl group; orproteins are not subjected to modification before the immobilizationprocess.
 7. The method according to claim 2, wherein the immobilizationof proteins is performed by sulfhydryl groups in a polymer carrier. 8.The method according to claim 2, wherein the immobilization of proteinsis performed by amino-groups in a polymer carrier.
 9. The methodaccording to claim 2, wherein the immobilization of proteins isperformed by aminoacids in a polymer carrier.
 10. The method accordingto claim 6, wherein the immobilization of oligonucleotides is performedby 5′-terminal of the oligonucleotide.
 11. The method according to claim6, wherein the immobilization of oligonucleotides is performed by3′-terminal of the oligonucleotide.
 12. The method according to claim 1,wherein a polymer layer is formed and is covalently bonded to asubstrate.
 13. The method according to claim 1, wherein a polymer layeris formed and is not covalently bonded to a substrate.
 14. The methodaccording to claim 1, wherein a polymer layer is formed on a substrateis a three-dimensional gel.
 15. The method according to claim 1, whereina polymer layer is formed on a substrate and is a compact continuouslayer.
 16. The method according to claim 1, wherein polymer layer isformed on a substrate and is divided by empty spaces into several cellsand each cell may comprise or not comprise an oligonucleotide, nucleicacid, and/or protein, and said oligonucleotide, nucleic acid, and/orprotein being immobilized in various cells may have different nature andproperties.
 17. The method according to claim 16, wherein said cellsform a phase being a one- or two-dimensional structure.
 18. The methodaccording to claim 1, wherein an application of the polymerizationmixture on a substrate is carried out by using an automatic deviceequipped with one or more microdispensers.
 19. The method according toclaim 18, wherein said microdispensers are pin type microdispensers. 20.The method according to claim 18, wherein said microdispensers aremicrodispensers of a jet type.
 21. The method according to claim 16,wherein before polymerization, one or more substrates including dropletsof composition are held in a sealed container comprising the samecomposition mixture in amounts more than two times exceeding the totalamount of the polymerization mixture as supported on substrates.
 22. Themethod according to claim 16, wherein one or more substrates includingsupported droplets of composition, before and during polymerization, areplaced into a sealed container under oxygen free inert atmosphere with acontrolled humidity.
 23. The method according to claim 22, wherein oneor more substrates including supported droplets of composition, beforeand during polymerization, are placed into a sealed container which isfilled with a gas selected from the group consisting of N₂, Ar, and CO₂.24. The method according to claim 23, wherein said gas is continuouslyor periodically restored in the container.
 25. The method according toclaim 2, wherein E is selected from the group consisting of water;N,N-dimethylformamide and dimethylsulfoxide.